The ultimate goal of this proposal is the identification of compounds which inhibit the function(s) of the human immunodeficiency virus (HIV) accessory protein Nef. In vivo experiments in rhesus monkeys indicate that the simian immunodeficiency virus (SIV) nef is critical pathogenic determinant of this virus. It has recently been shown that CD4 down- regulation is a common property of HIV-1 nef genes isolated directly from peripheral blood lymphocytes of HIV-1 infected individuals. In addition, Nef protein from the HIV-1 NL43 isolate has a dramatic effect on the development of CD4+ T cells when expressed in transgenic mice and this effect correlates with CD4 down-regulation in thymic T cells. Stable human T lymphoid cells expressing this Nef allele display block in interleukin 2 (IL-2) and NF-kappaB T cell receptor-mediated transcriptional activation. Since blocking IL-2 activation results in immunosuppression, this observation represents a potentially important functional property of Nef. This Nef property has not yet been correlated with CD4 down-regulation, nor has it been analyzed using other Nef isolates. We propose to transiently express several HIV-1 and HIV-2 primary Nef isolates together with an IL-2 promoter/luciferase and NF- kappaB/luciferase reporter constructs, in human T cell lines, and investigate whether the ability to impair the IL-2 and NF-kappaB transcriptional induction in response to stimuli that mimic T cell activation is a general property of functional Nef alleles. Promoter/luciferase reporter assays represent an excellent system for the accurate, automated screening of large number of compounds. If a block in IL-2 induction is a common property of primary Nef isolates, we will establish a cell-based system to search for compounds which inhibit the Nef-mediated block in IL-2 induction, using the unique high-throughput screening system developed at Oncogene Science.